JP5114702B2 - Method and apparatus for forming bilayer film by contact with amphiphilic monolayer - Google Patents

Description

  The present invention relates to a method and apparatus for forming a bilayer membrane for membrane protein analysis used in the fields of biotechnology, biochip, membrane protein analysis, drug discovery screening, biosensor, and the like.

Membrane proteins are present in cell membranes and play an important role in immune reactions and transport and excretion of substances inside and outside the cell. Therefore, elucidating the functions and characteristics of various membrane proteins one by one is the next generation. It has become an important issue for the development of new treatments and drug discovery methods.
Ion channels such as typical conventional methods for planar lipid membranes prepared for membrane protein analysis plane film method, i.e., brush coating method and LB method (L a ngmuir-Blodgett method). In both methods, a lipid bilayer is formed in a small pore of about several hundred microns opened in a Teflon (registered trademark) sheet or the like in a chamber filled with a buffer. The latter method is to apply a planar lipid membrane by gradually raising the solution surface of the chambers on both sides of the Teflon (registered trademark) sheet, utilizing the fact that a monolayer of lipid is formed on the liquid surface. It is a method of forming.

FIG. 14 is a schematic diagram showing such a conventional method for forming a planar lipid bilayer by the LB method.
In this figure, 101 is a Teflon (registered trademark) sheet, 102 is a small hole opened in the Teflon (registered trademark) sheet 101, 103 is a solution in which a monolayer of lipid 104 is formed on the surface, and 105 is a buffer solution. , and the have to form a lipid bilayer 106 by gradually increasing the Teflon on both sides of Chang Ba solution 103 surface of the sheet 101.

In addition, as a method of forming a lipid bilayer,
(1) A method of forming a lipid bilayer by developing amphiphilic molecules at the interface of a liquid three-layer system and removing the liquid in the intermediate layer (see Patent Document 1 below)
(2) After bringing one surface of the substrate having a small pore penetrated into contact with the surface of the first aqueous solution, a solution containing a lipid bilayer-forming molecule is added onto the first aqueous solution in the small pore. Furthermore, a method of supplying a second aqueous solution onto the other surface of the substrate (see Patent Document 2 below) has been proposed.
Japanese Patent Laid-Open No. 03-118832 JP 05-261277 A “Ion Channel Reconfiguration,” C.I. Miller Ed. , Plenum press, (1986) H. Suzuki, K .; Tabata, Y. et al. Kato-Yamada, H .; Noji, and S.J. Takeuchi, μTAS, 2,246 (2004) H. Suzuki et al, 3rd Int. IEEE-EMBS Special Topic Conf. on Microtechnologies in Medicine and Biology, Hawaii. May 2005. T.A. Ide, Y .; Takeuchi, and T.K. Yanagida, Single Moleculars. 3, 33 (2002).

However, both methods of the above-mentioned brushing and LB method, requires a large Chang bus of several cm, a dead volume is large, it is impossible microscopy. In the case of the conventional method (1), there is a difficulty in the process of removing the liquid in the intermediate layer. Furthermore, in the case of the method (2), a process for evaporation of the solution and a process for natural thinning are required, and it takes a long time to complete the process, and reproducibility is difficult.

Thus, the conventional method has a problem in forming a lipid bilayer membrane simply and accurately.
In view of the above problems, an object of the present invention is to provide a method for forming a bilayer film by contact with an amphiphilic monolayer film that can be easily and accurately formed, and an apparatus therefor. To do.

In order to achieve the above object, the present invention provides
[1] A method of forming a bilayer membrane by contact of amphipathic monolayers, the chamber formed in the substrate, the organic solvent is divided into a plurality of organic solvent and the organic solvent containing amphipathic molecules by introducing immiscible liquid and, the organic solvent and surfactant to form a plurality of amphipathic monolayers with the liquid body, on both sides at the interface facing with the organic solvent and the liquid in the chamber A bimolecular film is formed by applying pressure to the plurality of amphiphilic monomolecular films to contact each other.

[2] In the method for forming a bimolecular film by contacting the amphiphilic monomolecular film as described in [1] above, the chamber has a substantially cross shape including first and second microchannels formed orthogonal to each other. A chamber, introducing the liquid into the second microchannel, introducing the organic solvent into the first microchannel, and the liquid at an intersection of the first and second microchannels. wherein the interface facing the organic solvent and the liquid to form the amphipathic monolayer pressure of the liquid is controlled by a syringe pump by separating the pre SL both amphiphilic monolayers engaged fusion in contact with each other and forming a bilayer membrane.

[3] In the method for forming a bilayer membrane by contact with an amphiphilic monolayer according to [1] above, the chamber is a chamber that is divided into a plurality of compartments by a constricted portion, and an upper surface is opened, Filling the chamber with the organic solvent, dropping the liquid droplets for each of the plurality of compartments to form the amphiphilic monolayer at the interface between the organic solvent and each of the liquid droplets, By controlling the size of the droplet, the amphiphilic monolayer is brought into contact with each other and fused to form a bilayer.

[4] In the method for forming a bilayer membrane by contact with an amphiphilic monolayer according to [1] above, the chamber is divided into a plurality of compartments by a constricted portion, and the upper surface is closed, Filling the chamber with the organic solvent, introducing the liquid droplets into each of the plurality of compartments, forming the amphiphilic monolayer at the interface between the organic solvent and each of the liquid droplets, and a syringe The amphiphilic monolayer is brought into contact with each other and fused to form a bilayer by controlling the size of the droplets by a pump.

[5] The method for forming a bimolecular film by contact with an amphiphilic monomolecular film according to any one of [1] to [4], wherein the liquid is an aqueous solution.
[6] The method for forming a bilayer membrane by contacting the amphiphilic monolayer membrane according to [3] or [4] above, wherein the plurality of compartments are arranged in a horizontal array.
[7] In the method for forming a bilayer membrane by contacting the amphiphilic monolayer membrane according to [3] or [4] above, the plurality of compartments are three compartments, and the outer shape of the chamber is substantially triangular. It is characterized by making.

[8] In the method for forming a bilayer membrane by contact with an amphiphilic monolayer according to [3] or [4] above, the plurality of compartments are five compartments, and the outer shape of the chamber is a substantially square shape It is characterized by making.
[ 9 ] In an apparatus for forming a bimolecular film by contact with an amphiphilic monomolecular film, a chamber formed in a substrate, an organic solvent containing amphiphilic molecules introduced into the chamber, and the chamber are introduced into the chamber. a liquid immiscible with the previous SL organic solvent that will be divided into a plurality in the organic solvent, the the interface between the organic solvent and the liquid to form a plurality of amphiphilic monolayers, between the liquid and the organic solvent and control means for applying a pressure from both sides to the interface facing, and forming a bilayer in contact with each other by said control means said plurality of amphipathic monolayers.

[ 10 ] In the apparatus for forming a bilayer membrane by contact with an amphiphilic monolayer as described in [ 9 ] above, the chamber is substantially cross-shaped including first and second microchannels formed orthogonal to each other A chamber, introducing the liquid into the second microchannel, introducing the organic solvent into the first microchannel, and the liquid at an intersection of the first and second microchannels. The amphiphilic monomolecular film is formed at the facing interface between the organic solvent and the liquid by separating the liquid, and the pressure of the liquid is controlled by a syringe pump to form the facing amphiphilic monomolecular film. It is characterized by forming a bilayer by contacting and fusing each other .

[11] In the apparatus for forming a bilayer membrane by contact with an amphiphilic monolayer according to [9] above, the chamber is divided into a plurality of compartments by a constricted portion, and the chamber has an open upper surface, Filling the chamber with the organic solvent, dropping the liquid droplets for each of the plurality of compartments to form the amphiphilic monolayer at the interface between the organic solvent and each of the liquid droplets, By controlling the size of the droplet, the amphiphilic monolayer is brought into contact with each other and fused to form a bilayer.

[12] In the apparatus for forming a bilayer membrane by contact with an amphiphilic monolayer according to [9] above, the chamber is divided into a plurality of compartments by a constricted portion, and the upper surface is closed, Filling the chamber with the organic solvent, introducing the liquid droplets into each of the plurality of compartments, forming the amphiphilic monolayer at the interface between the organic solvent and each of the liquid droplets, and a syringe The amphiphilic monolayer is brought into contact with each other and fused to form a bilayer by controlling the size of the droplets by a pump.

[ 13 ] The apparatus for forming a bilayer film by contact with an amphiphilic monolayer according to any one of [ 9 ] to [ 12 ], wherein the liquid is an aqueous solution.
[ 14 ] The apparatus for forming a bilayer membrane by contact with an amphiphilic monolayer membrane according to [ 11 ] or [ 12 ], wherein the plurality of compartments are arranged in a horizontal array .
[15] In the above [11] or [12] forming apparatus bilayer membrane by contact of amphipathic monolayers, wherein the plurality of compartments are three pieces of compartments der, the outer shape of the chamber is substantially triangular It is characterized by having a shape .

[ 16 ] In the method for forming a bilayer film by contact with an amphiphilic monolayer as described in [ 11 ] or [ 12 ] above, the plurality of compartments are 5 compartments, and the outer shape of the chamber is substantially rectangular. It is characterized by making.

According to the present invention, the following effects can be achieved.
(1) A simple and accurate bilayer can be formed.
(2) In the inventions according to claims 2, 4, 10 and 12 , the pressure and volume of the liquid can be controlled by controlling the syringe pump, which makes it possible to control the film thickness and dramatically improve reproducibility. Can be improved.

According to the method of forming a bilayer membrane by contact of amphipathic monolayers of the present invention, the chamber formed in board, is divided into a plurality of organic solvent and the organic solvent containing amphipathic molecules introducing a liquid immiscible with the organic solvent, the organic solvent and surfactant to form a plurality of amphipathic monolayers with the liquid body, opposed to the said organic solvent in said chamber liquid A bimolecular film is formed by applying pressure to the interface from both sides to bring the plurality of amphiphilic monomolecular films into contact with each other .

Hereinafter, embodiments of the present invention will be described in detail.
FIG. 1 is a diagram showing a method for forming a lipid bilayer by contacting a monolayer using the substantially cross-shaped chamber according to the first embodiment of the present invention, and FIG. FIG. 1B is a diagram illustrating a state in which aqueous solution interfaces are in contact with each other , and FIG . 1C is a diagram illustrating a state in which a membrane protein is incorporated in a lipid bilayer structure .

First, First, as shown in FIG. 1 (a), the liquid in the second microchannel 3 formed on a substrate 5 having a front and back (e.g., aqueous solution) 4 introducing. Next, an organic solvent 2 containing lipid molecules is introduced into the first microchannel 1 formed so as to be orthogonal to the second microchannel 3 . Since the organic solvent 2 is not mixed with the liquid (aqueous solution) 4, the liquid (aqueous solution) 4 is separated by the organic solvent 2 at the intersection of the first and second microchannels 1 and 3 . Syringe pumps (not shown) are arranged at both ends of the second microchannel 3, and the pressure of the liquid 4 in the second microchannel 3 can be controlled by adjusting the syringe pump. In this case, a KCl solution is used as the liquid (aqueous solution) 4 for energization. However, as long as it is only for the formation of a lipid bilayer, it is not mixed with the organic solvent 2 containing lipid molecules. A general aqueous solution can be used.

As described above, when the liquid 4 is introduced into the second microchannel 3 and the organic solvent 2 containing lipid molecules is introduced into the first microchannel 1 , as shown in FIG. A lipid monomolecular film 2A is formed at the interface between the liquid 4 and the organic solvent 2 at the intersection of the chamber 6 where the first microchannel 1 and the second microchannel 3 intersect.
Next, the surfaces of the two aqueous solutions 4 are brought close to each other by controlling the pressure by injecting the liquid 4 from the syringe pump. As shown in FIG. 1B, when the distance between the two lipid monolayers 2A is several angstroms or less, the lipid monolayer 2A is fused by the van der Waals attractive force to form a lipid bilayer 2B.

As shown in FIG. 1 (c), membrane protein 9 can be incorporated into the lipid bilayer structure of FIG. 1 (b) in order to conduct membrane transport experiments. In FIG. 1, 7 is an Ag / AgCl electrode, and 8 is a current / voltage measuring device.
FIG. 2 is a schematic view of an apparatus for forming a lipid bilayer membrane by contacting a monolayer according to the first embodiment of the present invention.

Cruciform chamber 6 is machined by the CAD / CAM modeling system on acrylic plastic (PMMA) substrate 5, to close the flow path, Ru paste Tei a thin PMMA plate substrate 5. Width and depth of the channel are both 0.5 mm. The aqueous solution 4 is introduced from two opposing inlets of the second microchannel 3, and the organic solvent 2 containing lipid molecules is introduced from the inlet provided at one end of the first microchannel 1. . Here, a gramicidin peptide ion channel, which is a kind of membrane protein, is used for ion molecule transport experiments. In order to detect the ion flow (current), the Ag / AgCl electrode 7 connected to the current / voltage measuring device 8 is connected to the flow path of two aqueous solutions (here, KCl solutions) 4 in the second micro flow path 3. Insert into. As described above, the injection and extrusion of the KCl solution 4 are controlled by the syringe pump (not shown). Thereby, the pressure and volume of the KCl solution 4 can be arbitrarily controlled.

Note that the organic solvent 2 containing lipid molecules used here, 1, 2-Dioleoyl-sn -Glycero-3-Phosphocholine (DOPC, CAS No; 4235-95-4) chloroform-hexadecane (1: 1 vol% ). In this experiment, the above DOPC was used at 25 mg / ml . Optimum conditions concentration is 0.5~25mg / m l. Examples of membrane components capable of forming a membrane include lipids (eg, lipids such as phosphatidylcholine), amphiphilic molecules, and surfactants.

FIG. 3 is a view showing a microscopic image when the aqueous solution interfaces are in contact with each other, showing the first embodiment of the present invention. The two aqueous solutions (KCl solution) 4 do not fuse with each other because lipid molecules exist on the surface. One of the advantages of the present invention is that even if the lipid bilayer is broken, the procedure can be easily and quickly started again by flowing the solvent in the chamber.

By the way, since the lipid bimolecular film is a thin dielectric film, it acts as a capacitor.
FIG. 4 is a diagram showing a capacitive transient current passing through the interface when a 10 mV rectangular signal according to the present invention is applied. In the figure, a indicates a capacitive transient current in a lipid bilayer membrane formed by contacting lipid monolayers on the surface of an aqueous solution, and b indicates a capacitive transient current before the contact.

  As can be seen from FIG. 4, when a rectangular wave voltage of 100 mV (100 Hz) is applied, film capacitance characteristics are generated by forming a thin film. In this experimental result, the membrane capacitance corresponding to 130 pF was measured when the aqueous solution interfaces were in contact with each other (a in the figure). This confirmed that a lipid bilayer membrane structure was formed at the interface between the aqueous solutions in contact.

Next, in order to further confirm that the lipid membrane formed above is a bilayer membrane, a gramicidin peptide ion channel that opens in synchronism with the dimerization of the monomer of each monolayer membrane is introduced into the aqueous solution.
FIG. 5 is a diagram showing a time-series change in current passing through a lipid bilayer membrane incorporating gramicidin when 63 mV is applied according to an embodiment of the present invention.

First, lipid monolayers are brought into contact with each other [FIG. 5 (a)]. When the surface of the lipid monolayer is kept in contact, the current increases corresponding to the enlargement of the lipid bilayer region [FIG. 5 (b)]. Next, we separate the lipid monolayer surface from each other under the control of the syringe pump [FIG 5 (c), (d)] and the current drops to a reference value (gramicidin concentration in 100mM of KCl 5 0.0 × 10 ⁻¹² M).

Thus, after the aqueous solution interface comes into contact, the flow of K ⁺ ions (current) gradually increases, which corresponds to the enlargement of the lipid bilayer region. On the other hand, when the interface is separated, the current drops to zero. Gramicidin conducts current only when it is in the bilayer membrane, and this result shows that a lipid bilayer membrane is present during contact at the aqueous solution interface.
As described above, in the present invention, a lipid monomolecular film that contacts the microfluidic chip has been created for reconstitution of the lipid bimolecular film in the vertical direction. A lipid bilayer membrane made from two lipid monolayers according to the present invention showed a capacitance consistent with the conventional BLM method, and a gramicidin channel was formed in the membrane. Therefore, it was confirmed that the membrane formed by the present invention was a lipid bilayer membrane. Here, the membrane transport was electrically monitored using an ion channel. This technique also enables fluorescence imaging of molecular transport across the membrane.

FIG. 6 is a schematic plan view of a device for forming a lipid bilayer membrane by contacting monomolecular membranes having a plurality of chambers having a constricted portion according to a second embodiment of the present invention, and FIG. 7 is a lipid bilayer membrane using the same. It is a schematic perspective view which shows the formation method.
First, as shown in FIG. 6 (a) or FIG. 7 (a), on the base plate 11 to form a separated chamber 12 in constriction 13 whose upper surface is opened. Next, as shown in FIG. 6B or FIG. 7B, the chamber 12 having the constricted portion 13 is filled with an organic solvent 14 containing lipid molecules. Next, FIG. 6 (c), or as shown in FIG. 7 (c), constricted portions each separated by the chamber 12 at 13, an aqueous solution of the droplets 16, 17 which is immiscible in the organic solvent 14 containing lipid molecules Is dropped with a pipette 15 (see FIG. 7C). Then, in this state, a lipid monomolecular film 18 is formed at the interface between the aqueous solution droplets 16 and 17 and the organic solvent 14 containing lipid molecules. Next, as shown in FIG. 6 (d) or FIG. 7 (d), the aqueous solution droplets 16 and 17 are enlarged (aqueous solution droplets 16 ′ and 17 ′), and the aqueous solution droplets 16 ′ and 17 ′. , The lipid monolayer 18 formed at the interface between the aqueous solution droplets 16 ′ and 17 ′ and the organic solvent 14 containing lipid molecules also contacts and fuses to form a lipid bilayer 19. Is done.

FIG. 8 is a schematic view of an apparatus for forming a lipid bilayer membrane by contacting monolayers having a plurality of chambers having a constricted portion showing a modification of the second embodiment of the present invention.
In this embodiment, on the base plate 21 to form a chamber 22 having a constricted portion 23 whose upper surface is closed, the flow path 25 for sending the organic solvent 24 containing lipid molecules in the chamber 22, Ji Yanba 22 each A flow path 26 for introducing the aqueous solution droplet 27, a syringe pump 28 disposed in the flow path 26, and a control device 29 connected to the syringe pump 28 are provided.

Therefore, an organic solvent 2 4 from the channel 25 containing lipid molecules is introduced into the chamber 22, then, the droplets 27 of the aqueous solution immiscible and organic solvent 24 containing lipid molecules from the flow passage 26 is introduced. Then, the aqueous solution droplet 27 is controlled by the control of the syringe pump 28, and the lipid monomolecular film formed at the interface between the aqueous solution droplet 27 and the organic solvent 24 containing lipid molecules comes into contact and fuses to form a lipid. A bimolecular film 24B is formed.

9 and 10 are experimental examples of the present invention, and FIG. 9 is a diagram showing a structure of a chip device having a plurality of chambers having a constricted portion of the experimental example of the present invention, and FIG. overall top view, and FIG. 9 (b) is an enlarged plan view of the multiple chamber section, FIG. 9 (c) a side surface cross-sectional view of Waso, Fig 9 (d) are drawing-substituting photograph showing a perspective view of the entire It is.
9A, the length x of the substrate 30 is 30 mm and the width y is 20 mm. In FIG. 9B, the radius r of the chamber 31 is 2 mm, the length l between the constricted portions 32 is 2 mm, and In FIG. 9C, the thickness d of the substrate 30 is 2 mm. Further, the material of the substrate 30 is acrylic.

FIG. 10 is a diagram showing a method for forming a lipid bilayer membrane according to an experimental example of the present invention.
(1) First, the organic solvent (15 μl) 33 containing lipid molecules is filled in the chamber 31 shown in FIG. 9 [FIG. 10 (a-1), which is a perspective view of the chip device, and is a side sectional view of the chip device. 10 (a-2) and FIG. 10 (a-3) which is a top view of the chip device, respectively].

(2) Next, the first droplet (15 μl) 35 of the aqueous solution not mixed with the organic solvent 33 containing lipid molecules is introduced into the organic solvent 33 containing lipid molecules by the pipette 34 [side cross-section of chip device FIG. 10B-1 is a diagram, and FIG. 10B-2 is a top view of the chip device.
(3) Next, a second droplet (15 μl) 36 of an aqueous solution not mixed with the organic solvent 33 containing lipid molecules is introduced [FIG. 10 (c-1), which is a side sectional view of the chip device, chip See FIG. 10 (c-2), which is a top view of the device, respectively.

(4) Then, as shown in FIG. 10 (d), in the constricted portion 32 (see FIG. 9) of the chip device, a lipid bilayer membrane 38 is obtained at the contact portion 37 of the aqueous solution droplets 35, 36.
Figure 11 is a third embodiment comprising a Chang bar separated into three horizontal arrangement by two constricted portions showing a schematic plan of the forming apparatus of the lipid bilayer to form a two lipid bilayer of the present invention FIG.

In this embodiment, a chamber 42 having two laterally constricted portions 43 and 44 formed on a substrate 41 is prepared. First, an organic solvent 45 containing lipid molecules is introduced into the chamber 42, and then there. by introducing three droplets 46, 47, 48, the first lipid bilayer 49 on the contact surface thereof with the droplets 46 and 47, the the contact surface with their droplet 47 and 48 Second lipid bilayer membranes 50 are respectively formed.

FIG. 12 is a schematic plan view of a lipid bilayer membrane forming apparatus having a chamber having a substantially triangular outer shape for forming three lipid bilayer membranes according to a fourth embodiment of the present invention.
In this embodiment, the chamber 51 has a substantially triangular shape, and by introducing three droplets 53, 54, 55 into an organic solvent 52 containing lipid molecules, the contact surface between the droplets 53 and 54 is introduced. The first lipid bilayer 56, the second lipid bilayer 57 on the contact surface of the droplets 54 and 55, and the third lipid bilayer 58 on the contact surface of the droplets 55 and 53, respectively. be able to.

FIG. 13 is a schematic plan view of an apparatus for forming a lipid bilayer membrane having a chamber having a substantially rectangular outer shape for forming four lipid bilayer membranes according to a fifth embodiment of the present invention.
In this embodiment, the chamber 61 has a substantially rectangular shape, and five droplets 63, 64, 65, 66, 67 are introduced into an organic solvent 62 containing lipid molecules. In other words, the droplet 63 is located in the center, by Rukoto droplets 64, 65, 66, 67 are arranged therearound, the first lipid bilayer 68 the contact surface between the droplet 63 and 64, the liquid The second lipid bilayer 69 on the contact surface of the droplets 63 and 65, the third lipid bilayer 70 on the contact surface of the droplets 63 and 66, and the fourth lipid on the contact surface of the droplets 63 and 67. Each of the lipid bilayers 71 can be formed.

The shape of the chamber is not limited to the one described above. As described above, a lipid monomolecular film is formed at the interface between the droplet and the organic solvent containing lipid molecules, and the lipid bilayer is formed at the constricted portion of the chamber. If a molecular film can be formed, it can be transformed into various shapes.
In this way, a lipid monomolecular film is formed by introducing a droplet of an aqueous solution that is not mixed with an organic solvent into an organic solvent containing lipid molecules in advance and controlling the droplet. A lipid bilayer membrane can be formed by contacting and fusing.

In the above embodiment, the control of the liquid that is not mixed with the organic solvent exclusively containing lipid molecules has been described. However, the organic solvent containing lipid molecules may be controlled. For example, by reducing the pressure (volume) of the organic solvent containing lipid molecules, a plurality of lipid monolayers may be brought into contact and fused to form a lipid bilayer.
Moreover, although the Example using the organic solvent containing a lipid molecule was shown in the said Example, you may make it use the organic solvent containing an amphiphilic molecule | numerator. Examples of the amphiphilic molecules include phospholipids, long-chain alcohols, long-chain cambonic acids, soaps, synthetic detergents, and the like. These amphiphilic molecules include nonpolar solution / aqueous solution interfaces and gas phase / aqueous solutions. A monomolecular film can be formed at the interface.

  In addition, this invention is not limited to the said Example, Based on the meaning of this invention, a various deformation | transformation is possible and these are not excluded from the scope of the present invention.

  Membrane proteins play an important role in physiological functions such as drug response, energy conversion, immune reaction, mass transport, and information transmission. Many of the membrane proteins are main targets for drug discovery, and the market size of drugs related to a series of receptor proteins called GPCR (G-protein coupled receptor; G protein coupled receptor) is large. For this reason, it is expected that membrane proteins are arrayed on a chip, but there has been no report of efficiently reconstituting a lipid planar membrane on an array. In addition, there is no device that can measure the membrane current as in the physiological condition setting.

  For example, in the Human Genome Project, all GPCR genes have already been identified, and the number of targets that can be practically limited is limited. Therefore, there is an urgent need to arrange these in an array on the chip and examine the response to the drug for each GPCR. Besides these, membrane proteins are also the main next-generation drug targets, such as a series of membrane proteins called ABC transporters that are responsible for drug resistance of cancer cells. The development of this system can contribute to rapid drug development by incorporating these drug discovery target membrane proteins.

It is a figure which shows the lipid bilayer membrane formation method by the contact of a monolayer using the substantially cross-shaped chamber which shows 1st Example of this invention. It is a schematic diagram of the formation apparatus of the lipid bilayer membrane by the contact of the monomolecular membrane which shows 1st Example of this invention. It is a figure which shows the microscope image when the aqueous-solution interfaces are contacting, which shows 1st Example of this invention. It is a figure which shows the capacitive transient current (capacitive transient current) which passes the interface when a 10 mV rectangular signal concerning this invention is applied. It is a figure which shows the time-sequential change of the electric current which passes the lipid bilayer membrane which incorporated gramicidin when 63 mV which shows the Example of this invention is applied. It is a schematic plan view of the lipid bilayer membrane formation apparatus by the contact of the monomolecular membrane provided with the some chamber which has a constriction part which shows 2nd Example of this invention. It is a schematic perspective view which shows the lipid bilayer membrane formation method by the contact of a monomolecular membrane provided with the several chamber which has a constriction part which shows 2nd Example of this invention. It is a schematic diagram of the lipid bilayer membrane formation apparatus by the contact of the monomolecular membrane provided with the some chamber which has the narrow part which shows the modification of 2nd Example of this invention. It is a figure which shows the structure of the chip device provided with the several chamber which has the constriction part of the experiment example of this invention. It is a figure which shows the formation method of the lipid bilayer membrane of the experiment example of this invention. Is the third with a Chang bar separated into three horizontal arrangement by two constricted portion showing an embodiment, a schematic plan view of a forming apparatus of the formed lipid bilayer of two lipid bilayer of the present invention . It is a plane schematic diagram of the apparatus for forming a lipid bilayer membrane having a chamber having a substantially triangular outer shape for forming three lipid bilayer membranes according to the fourth embodiment of the present invention. FIG. 10 is a schematic plan view of a lipid bilayer forming apparatus having a chamber having a substantially quadrangular outer shape for forming four lipid bilayers according to a fifth embodiment of the present invention. It is a schematic diagram which shows the planar lipid bilayer membrane formation method by the conventional LB method.

DESCRIPTION OF SYMBOLS 1 1st microchannel 2, 14, 24, 33, 45, 52, 62 Organic solvent containing lipid molecule 2A, 18 Lipid monomolecular film 2B, 19, 24B, 38 Lipid bimolecular film 3 2nd micro flow Path 4 Liquid not mixed with organic solvent containing lipid molecules (eg, aqueous solution)
5, 11, 21, 30 and 41 groups plate <br/> 6 cross-shaped chamber 7 Ag / AgCl electrode 8 current and voltage measuring device
Nine membrane proteins 12, 22, 31, 42, 51, 61 Chamber 13, 23, 32, 43, 44 Constricted part 15, 34 Pipette 16, 17, 16 ', 17', 27, 46, 47, 48 , 53, 54, 55, 63, 64, 65, 66, 67 Droplet of aqueous solution not mixed with organic solvent containing lipid molecule 25, 26 Flow path 28 Syringe pump 29 Control device 35 First droplet of aqueous solution 36 Second droplet of aqueous solution 37 Contact portion of droplet of aqueous solution 49, 56, 68 First lipid bilayer membrane 50, 57, 69 Second lipid bilayer membrane 58, 70 Third lipid bimolecule Membrane 71 Fourth lipid bilayer membrane

Claims (16)

Interface to the chamber formed substrate, by introducing a liquid immiscible with the organic solvent which is divided into a plurality of organic solvents and organic solvent containing amphipathic molecules, and the organic solvent and the liquid body Forming a plurality of amphiphilic monolayers, and applying pressure from both sides to the facing interface between the organic solvent and the liquid in the chamber to bring the plurality of amphipathic monolayers into contact with each other. A method for forming a bilayer film by contacting an amphiphilic monolayer film, comprising forming a molecular film. The method for forming a bilayer membrane by contact with an amphiphilic monolayer according to claim 1, wherein the chamber is a substantially cruciform chamber including first and second microchannels formed orthogonal to each other, Introducing the liquid into the second microchannel, introducing the organic solvent into the first microchannel, and separating the liquid at an intersection of the first and second microchannels; wherein the interface facing the organic solvent and the liquid to form the amphipathic monolayer pressure of the liquid is controlled by a syringe pump, by contacting the pre-Symbol both amphiphilic monolayers together by method of forming a bilayer membrane by contact of amphipathic monolayers and forming a bilayer membrane is engaged fusion. 2. The method for forming a bilayer film by contact with an amphiphilic monolayer according to claim 1, wherein the chamber is a chamber that is divided into a plurality of compartments by a constricted portion and an upper surface is opened, and the organic layer is placed in the chamber. Filling a solvent, dropping the liquid droplets for each of the plurality of compartments to form the amphiphilic monolayer at the interface between the organic solvent and each of the liquid droplets. A method of forming a bilayer by contact of an amphiphilic monolayer, wherein the bilayer is formed by bringing the amphipathic monolayer into contact with each other and fusing them by controlling the thickness. 2. The method of forming a bilayer membrane by contacting an amphiphilic monolayer according to claim 1, wherein the chamber is a chamber that is partitioned into a plurality of compartments by a constricted portion and the upper surface is closed, and the organic layer is placed in the chamber. Filling the solvent, introducing the liquid droplets into each of the plurality of compartments to form the amphiphilic monomolecular film at the interface between the organic solvent and each of the liquid droplets. A method for forming a bilayer film by contact with an amphiphilic monolayer, wherein the amphipathic monolayer is brought into contact with and fused with each other by controlling the size of a droplet. 5. The method for forming a bilayer film by contact with an amphiphilic monolayer according to claim 1, wherein the liquid is an aqueous solution. Method for forming a molecular film. 5. The method for forming a bilayer membrane by contact with an amphiphilic monolayer according to claim 3 or 4, wherein the plurality of compartments are arranged in a horizontal array. Method for forming a molecular film. 5. The method for forming a bilayer membrane by contact with an amphiphilic monolayer according to claim 3 or 4, wherein the plurality of compartments are three compartments, and the outer shape of the chamber is substantially triangular. A method for forming a bilayer film by contacting an amphiphilic monolayer film. The method for forming a bilayer membrane by contact with an amphiphilic monolayer according to claim 3 or 4, wherein the plurality of compartments are five compartments, and the outer shape of the chamber is a substantially square shape. A method for forming a bilayer film by contacting an amphiphilic monolayer film. (A) a chamber formed in the substrate;
(B) an organic solvent containing amphiphilic molecules introduced into the chamber;
(C) a liquid immiscible with the previous SL organic solvent that will be divided into a plurality in the organic solvent introduced into the chamber,
; (D) an organic solvent and surfactant to form a plurality of amphipathic monolayers with the liquid, and control means for applying a pressure from both sides to the interface facing with the said organic solvent liquid,
(E) forming the plurality of amphipathic monolayers a bilayer membrane by contact of the two amphiphilic monolayers you and forming a bilayer in contact with each other by the control unit device. The apparatus for forming a bilayer membrane by contact with an amphiphilic monolayer according to claim 9 , wherein the chamber is a substantially cruciform chamber including first and second microchannels formed orthogonal to each other, Introducing the liquid into the second microchannel, introducing the organic solvent into the first microchannel, and separating the liquid at an intersection of the first and second microchannels; The amphiphilic monomolecular film is formed at the facing interface between the organic solvent and the liquid by a syringe, and the pressure of the liquid is controlled by a syringe pump so that the facing amphiphilic monomolecular films are brought into contact with each other and fused. An apparatus for forming a bilayer film by contact with an amphiphilic monolayer film, characterized in that a bilayer film is formed. 10. The apparatus for forming a bilayer membrane by contact with an amphiphilic monolayer according to claim 9, wherein the chamber is a chamber that is partitioned into a plurality of compartments by a constricted portion, and an upper surface is opened, and the organic layer is placed in the chamber. Filling a solvent, dropping the liquid droplets for each of the plurality of compartments to form the amphiphilic monolayer at the interface between the organic solvent and each of the liquid droplets. A device for forming a bilayer film by contact with an amphiphilic monolayer, wherein the amphipathic monolayer is brought into contact with and fused to form a bilayer by controlling the thickness. 10. The apparatus for forming a bilayer membrane by contact with an amphiphilic monolayer according to claim 9, wherein the chamber is a chamber that is partitioned into a plurality of compartments by a constricted portion, and an upper surface is closed, and the organic layer is placed in the chamber. Filling the solvent, introducing the liquid droplets into each of the plurality of compartments to form the amphiphilic monomolecular film at the interface between the organic solvent and each of the liquid droplets. An apparatus for forming a bilayer by contact with an amphiphilic monolayer, wherein the amphipathic monolayer is brought into contact with each other and fused to form a bilayer by controlling the size of a droplet. 13. The apparatus for forming a bimolecular film by contact with an amphiphilic monomolecular film according to any one of claims 9 to 12 , wherein the liquid is an aqueous solution. Molecular film forming equipment. 13. The apparatus for forming a bilayer membrane by contact with an amphiphilic monolayer according to claim 11 or 12 , wherein the plurality of compartments are arranged in a horizontal array. Molecular film forming equipment. In forming apparatus bilayer membrane by contact of amphipathic monolayers of claim 11 or 12, wherein, wherein the plurality of compartments are three pieces of compartments der, the outer shape of the chamber form a substantially triangular shape An apparatus for forming a bilayer film by contact with an amphiphilic monolayer film. 13. The method of forming a bilayer membrane by contacting an amphiphilic monolayer according to claim 11 or 12 , wherein the plurality of compartments are five compartments, and the outer shape of the chamber is substantially rectangular. A device for forming a bilayer film by contacting an amphiphilic monolayer film.